Wu Yuanqing, Zhang Yuanyuan, Tu Ran, Liu Hao, Wang Qinhong
College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.
Sheng Wu Gong Cheng Xue Bao. 2013 Jun;29(6):760-71.
Promoter is one of important elements for gene expression and regulation. In the construction of recombinants for metabolic engineering and synthetic biology, it is necessary to have the promoters with varying strengths for fine-tuning metabolic pathway to reach the metabolic balance, decrease the accumulation of intermediate and increase the production of target metabolite. However, the natural promoters available are not completely suitable for fine-tuning metabolic pathway due to discrete strength, lack of versatility and standardization. To deal with this problem, in this study, a new 88 bp synthetic promoter, which contains the typical -35 box, -10 box as well as ribosome bind site, was designed. Then, the promoter library was constructed by introducing some degenerate base pairs in the sequence of 6 bp in the upstream of the initial transcription site and 14 bp in spacer region between -35 and -10 box. 720 promoters with varying strengths were screened out from a library of more than 5 000 clones via the expression of red fluorescent protein mCherry under the control of the synthetic promoter. The sequence analysis based on 35 promoters with varying strengths showed the promoters with varying strengths are base preference. The purine bases in -13 site and pyrimidine bases in the transcriptional initiation sequence are of high frequency; the purine and pyrimidine bases are of the similar frequency in the spacer sequence between -35 and -10 box in strong promoter. In the end, five characterized promoters with varying strengths were selected to tune the synthetic pathway of cis,cis-muconic acid in Escherichia coli. The results showed that the promoters with varying strengths can regulate the production of cis,cis-muconic acid and the accumulation of the intermediate catechol.
启动子是基因表达和调控的重要元件之一。在代谢工程和合成生物学重组体的构建中，需要具有不同强度的启动子来精细调节代谢途径，以实现代谢平衡、减少中间产物的积累并提高目标代谢产物的产量。然而，现有的天然启动子由于强度离散、缺乏通用性和标准化，并不完全适合精细调节代谢途径。为了解决这个问题，在本研究中，设计了一个新的88 bp合成启动子，它包含典型的-35框、-10框以及核糖体结合位点。然后，通过在初始转录位点上游6 bp序列和-35框与-10框之间的间隔区14 bp中引入一些简并碱基对，构建了启动子文库。通过在合成启动子控制下红色荧光蛋白mCherry的表达，从一个超过5000个克隆的文库中筛选出了720个具有不同强度的启动子。基于35个具有不同强度启动子的序列分析表明，具有不同强度的启动子存在碱基偏好。-13位点的嘌呤碱基和转录起始序列中的嘧啶碱基出现频率较高；在强启动子中，-35框与-10框之间的间隔序列中嘌呤和嘧啶碱基的频率相似。最后，选择了五个具有不同强度的特征启动子来调节大肠杆菌中顺,顺-粘康酸的合成途径。结果表明，具有不同强度的启动子可以调节顺,顺-粘康酸的产量和中间产物儿茶酚的积累。
